We investigated spectral holes burnt at 1.5 K into the origins of several tautomeric forms of mesoporphyrin IX-substituted horseradish peroxidase at pH 8 under pressures up to 2 MPa. From the pressure-induced lineshift the compressibility of the apoprotein could be determined. We found that the compressibility changed significantly when measured at different tautomer origins. It was concluded that there must be a correlation between the tautomer configurations of the chromophore and the actual structures of the apoprotein. As a consequence, specific conformational substates of the protein can be selected by optical selection of the associated tautomers.The solid-state physics of proteins is an intriguing field. Unlike crystals, proteins are finite systems, yet they have a smooth density of vibrational states which is Debye-like at sufficiently small energies (1). Like crystals, proteins are highly ordered (2). Yet disorder plays a very important role, too (3, 4). Disorder manifests itself in inhomogeneously broadened spectral lines, in nonexponential kinetics, in nonArrhenius-type activated processes, in a glass-like specific heat, in dielectric damping, etc. (5-8). Even from x-ray scattering experiments, it became obvious that, at a sufficiently high level of resolution, the structure of a protein is not so well defined (2, 3). There seems to be agreement that a certain extent of structural disorder is a prerequisite for the proper functioning of a protein.A possible model for describing the relation between order and disorder in proteins is based on the concept of conformational substates (4,7,9,10): The basic idea of this model is that a protein can exist in a huge set of substates. Most of these substates are assumed to be in fast equilibrium because their separating barriers are sufficiently small compared with kT. Some ofthem, however, are nonequilibrium states. These special substates may be functionally important. It is structural disorder which gives enough freedom to the protein to reorganize its structure for specific requirements.Here we address the problem of how this reorganization takes place and how this is related to the prosthetic group. Can a slight structural change of the prosthetic group, as induced by a weak chemical interaction, be a signal for the protein structure to be rearranged, for example, to help the binding of special molecules? That is, we address the problem of correlation between the configurations of the prosthetic group and the conformational substates of the protein.The experimental technique we employ is persistent spectral hole burning (11)(12)(13)(14)(15). Hole burning is a special type of saturation spectroscopy. Its specific feature, as compared with similar techniques in NMR and ESR spectroscopy, is the persistence of the hole. This persistence is the reason why the technique can be used to study the ground state by optical means. At liquid-helium temperatures, the width of a spectral hole is close to the natural width of the optical transition involved. For m...
Comparative spectral diffusion studies in the millikelvin range between a protein and a glass point to a strong shielding of the chromophore from the strain and/or electric fields of the host and to quite specific features of the energy landscape.
We performed hole-burning Stark effect experiments on cytochrome c in which the iron of the herne was either removed or replaced by Zn. According to the experiments, the free-base compound has an effective inversion center, even in the protein. The Zn compound, on the other hand, shows quite peculiar features: in the low-frequency range of the inhomogeneous band, it definitely has a dipole moment, as indicated by a splitting of the hole in the external field. However, in the maximum of the inhomogeneous band, a severe charge redistribution occurs, as the experiments show. In addition to the Stark experiments, we performed calculations of the electrostatic fields at the pyrrole rings and at the metal site of the heme group. We interpret our findings with a model based on structural hierarchies: the protein can exist in a few subconformations, which can be distinguished through the structure of the heme pocket. The different pocket structures support different structures of the chromophore, which, in turn, can be distinguished through their behavior in an external field. These distinct structures, in turn, correspond to a rather broad distribution of protein structures, which leave, however, the pocket structure largely unchanged. These structures show up in inhomogeneous broadening.
We measured the behavior of spectral· holes under isotropic pressure-changes as a function of bum frequency. We compared a protein sample, namely protoporphyrin IX substituted myoglobin in a glycerol/water glass with a sample where the protoporphyrin IX was directly dissolved in a host glass. The differences are remarkable-holes in the pure glass behave as expected for a homogeneous isotropic material. It is the nonlinear frequency dependence of the pressure shift where the deviation of the protein sample is most obvious. These observations signal a correlation between the structures of the dye probe and the structures of the apoprotein. They further show that global parameters of the apoprotein, such as the isothermal compressibility, depend strongly on the associated conformational substates and are subject to unexpected large variations.
We performed comparative Stark-effect experiments on spectral holes in a protein and a glass sample. The protein was protoporphyrin IX-substituted myoglobin in a glycerol/water solvent. The glass sample was a protoporphyrin IX-doped mixture of dimethylformamide/glycerol. As expected, in both cases the spectral holes varied linearly with the electric field. Yet, whereas in the protein the holes showed a clear splitting, they showed no splitting in the glass sample, irrespective of the chosen polarization of the laser. In both samples the hole broadened in the applied field. The magnitude of the broadening was about the same in both cases. The following conclusions were drawn. The absence of a splitting in the glass signals an effective global inversion symmetry of the chromophore, despite its low symmetry group. The dipole moment changes are random. In the protein the inversion symmetry is broken through the spatial correlation of the protein building blocks, leading to a molecular frame-fixed dipole moment difference and, hence, to the observed splitting. Despite these symmetry-breaking properties, the local structural randomness is of the same magnitude in the glass and in the protein, as is obvious from the broadening. The distinct difference in the Stark pattern shows that the range of the relevant chromophore interactions is confined to typical dimensions of the protein.The characterization of the solid state of proteins is not straightforward. Proteins do not fit into the usual categories of solid-state phases. They have many "in-between" properties. At sufficiently high temperatures, say above 200 K, they show a behavior in between those of liquids and crystals (1-10). At sufficiently low temperatures their properties are in between those of crystals and glasses. For instance, their specific heat at low temperature is glass-like (11,12), their dielectric as well as ultra-sound absorption is glass-like (11), and many of their optical as well as their Mossbauer properties are glass-like (2, 4, 5, 13-18). On the other hand, their x-ray diffraction pattern is well resolved and crystal-like (19). Yet, even the x-ray diffraction reveals glass-like properties when the DebyeWaller factor is analyzed (20, 21): the mean-square displacement (x2) averaged over the atoms of an amino acid residue varies along the backbone and, as the absolute temperature goes to zero, shows large deviations from the usual zero-point vibrational amplitudes. These findings show that there is an additional structural uncertainty in the positions of the protein building blocks which arises from a broad distribution of conformational substructures.In this paper we will show by using hole-burning Stark spectroscopy (22-27) that, on the one hand, the randomness in the structure of a protein is comparable to the randomness of a glass. Yet, on the other hand, there is a high spatial correlation in the protein which leads to a very specific Stark pattern. From this specific pattern we will draw an important conclusion as to the range of t...
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